ate appearance of phospholipid atelet-activating factor and I an skin after repeated antigen challen eir Shalit, MD,* Frank H. Valone, MD,** Paul C. Atkins, MD,* Warn D. Ratnoff, MD,*** Edward J. Goetzl, MD,*** and rton Zweiman, MD* Philadelphia, Pa., and San Francisco, Calif. bzjZammatory mediators were assessed in supernatants of chamber fluids from eight ragweed- or grass-sensitive subjects during antigen-induced cutaneous inflammatory responses. Platelet activating factor (PAP) accumulated at concentrations of 1 pm to 90 pmollL in six of eight subjects beginning at 3 hours and continuing for 9 hours after antigen challenge. Leukotriene B, (LTB,) was detectable at cutaneous sites of antigen challenge in jive of five subjects throughout the 9-hour period at levels from 1 to 36 nmol, a range of 38% to 80% of which were o-oxidation metabolites. Histamine levels peaked in the first hour at 106 ? 18 nglml atzd decreased to a plateau of I1 to 13 nglml at 3 to 9 hours after antigen challenge. No PAP und only very low levels of LTB, (0.1 to 1.3 nmol) and of histamine (<Z nglml) were detected at buffer-control sites during the 9 hours of study. Continuous antigen exposure thus results in the persistent release of histamine and LTB, and the late appearance of PAP, all of which may contribute to the chronic@ of allergic disorders and may have a bearing on the &E-mediated, late-phase cutaneous response. (J ALLERGY CLIN IMWNOL 1989;83:691-6.) We have previously used skin chambers placed over denuded skin blister sites to measure mediator release in ongoing allergic reactions in humans.’ In these ex- periments a single administration of antigen for 1 hour was followed by hourly replacement with buffer for 4 to 6 hours. Studies of mediator release have dem- onstrated the prolonged release of histamine, LTG, and activation of plasma kallikrein and Hageman fac- tor after antigen challenge, but not buffer challenge, of these denuded skin blister sites.lw3 However, no visible late-onset response occurred at these sites. More recently, prolonged administration of antigen for up to 5 hours enabled us to detect the beginning *Prom the Department of Medicine, Allergy and Immunology Sec- tion, University of Pennsylvania School of Medicine, Philadel- phia, Pa., **Department of Medicine, University of California Veterans Administration Medical Center, San Francisco, and ***Department of Medicine, Allergy and Immunology Section, University of California Medical Center, San Francisco, Calif. Supported by National Institutes of Health Grants AI 14332, AI 22141, AK 19784, and HL31809, and the Veterans Admin- istration. Received for publication March 25, 1988. Accepted for publication Aug. 27, 1988. Reprint requests: Paul C. Atkins, MD, Allergy and Immunology Section, 5th Floor Johnson Pavilion, Philadelphia, PA 19104. 6057. Abbreviations used LTC.,, LTB,: Leukotrienes C, and B, PAF: Platelet-activating factor PBS: Phosphate-buffered saline HPLC: High-performance liquid chromatog- raphy I RIA: Radioimmunoassay of a visible late-phase response at these sites, as well as to recover cells from the chamber fluids that were activated and functional when these were tested by different markers and assays.4s 5 We ~~de~ook these studies in eight highly sensitive atopic subjects to de- termine during the course of 7 to 9 hours, encom- passing the time period of maximal appearance of Iate- onset reactions in the skin, whether the lipid mediators PAF and LTB4 that can cause late-phase responses in humans were detectable in these chamber fluids. PAF and LTB, both are derived from distinct con- stituents of cellular phospholipids and are cap mediating leukocyte chemotaxis and other fu as well as other elements of in~a~at~o~.~, 7 Intra- dermal injection of PAF or LTB, in normal humans elicits an early wheal-and-flare response, followed by a cellular inflammatory reaction with a perivascular-
Transcript
ate appearance of phospholipid atelet-activating factor and I
an skin after repeated antigen challen
eir Shalit, MD,* Frank H. Valone, MD,** Paul C. Atkins, MD,* Warn D. Ratnoff, MD,*** Edward J. Goetzl, MD,*** and
rton Zweiman, MD* Philadelphia, Pa., and San Francisco, Calif.
bzjZammatory mediators were assessed in supernatants of chamber fluids from eight ragweed- or grass-sensitive subjects during antigen-induced cutaneous inflammatory responses. Platelet activating factor (PAP) accumulated at concentrations of 1 pm to 90 pmollL in six of eight subjects beginning at 3 hours and continuing for 9 hours after antigen challenge. Leukotriene B, (LTB,) was detectable at cutaneous sites of antigen challenge in jive of five subjects throughout the 9-hour period at levels from 1 to 36 nmol, a range of 38% to 80% of which were o-oxidation metabolites. Histamine levels peaked in the first hour at 106 ? 18 nglml atzd decreased to a plateau of I1 to 13 nglml at 3 to 9 hours after antigen challenge. No PAP und only very low levels of LTB, (0.1 to 1.3 nmol) and of histamine (<Z nglml) were detected at buffer-control sites during the 9 hours of study. Continuous antigen exposure thus results in the persistent release of histamine and LTB, and the late appearance of PAP, all of which may contribute to the chronic@ of allergic disorders and may have a bearing on the &E-mediated, late-phase cutaneous response. (J ALLERGY CLIN IMWNOL 1989;83:691-6.)
We have previously used skin chambers placed over denuded skin blister sites to measure mediator release in ongoing allergic reactions in humans.’ In these ex- periments a single administration of antigen for 1 hour was followed by hourly replacement with buffer for 4 to 6 hours. Studies of mediator release have dem- onstrated the prolonged release of histamine, LTG, and activation of plasma kallikrein and Hageman fac- tor after antigen challenge, but not buffer challenge, of these denuded skin blister sites.lw3 However, no visible late-onset response occurred at these sites. More recently, prolonged administration of antigen for up to 5 hours enabled us to detect the beginning
*Prom the Department of Medicine, Allergy and Immunology Sec- tion, University of Pennsylvania School of Medicine, Philadel- phia, Pa., **Department of Medicine, University of California Veterans Administration Medical Center, San Francisco, and ***Department of Medicine, Allergy and Immunology Section, University of California Medical Center, San Francisco, Calif.
Supported by National Institutes of Health Grants AI 14332, AI 22141, AK 19784, and HL31809, and the Veterans Admin- istration.
Received for publication March 25, 1988. Accepted for publication Aug. 27, 1988. Reprint requests: Paul C. Atkins, MD, Allergy and Immunology
Section, 5th Floor Johnson Pavilion, Philadelphia, PA 19104. 6057.
Abbreviations used LTC.,, LTB,: Leukotrienes C, and B,
of a visible late-phase response at these sites, as well as to recover cells from the chamber fluids that were activated and functional when these were tested by different markers and assays.4s 5 We ~~de~ook these studies in eight highly sensitive atopic subjects to de- termine during the course of 7 to 9 hours, encom- passing the time period of maximal appearance of Iate- onset reactions in the skin, whether the lipid mediators PAF and LTB4 that can cause late-phase responses in humans were detectable in these chamber fluids.
PAF and LTB, both are derived from distinct con- stituents of cellular phospholipids and are cap mediating leukocyte chemotaxis and other fu as well as other elements of in~a~at~o~.~, 7 Intra- dermal injection of PAF or LTB, in normal humans elicits an early wheal-and-flare response, followed by a cellular inflammatory reaction with a perivascular-
2 Shalit et a;.
leukocytic infiltrate composed predominantly of neutrophils ~‘3 9 High levels of PAF were found in blood of patients with primary-acquired cold urticaria after cold challenge and in the nasal secretions of allergic subjects after local challenge with specific antigen. lo, ” Analysis of the time course and antigen- concentration dependence of appearance of PAF and LTB, in chamber fluids over denuded blister bases of allergic subjects now have demonstrated that both lipid mediators attain peak concentrations late after continuous antigen challenge.
Eight healthy atopic volunteers (five male and three fe- male subjects, 20 to 33 years of age) with seasonal allergic rhinitis, skin sensitive ta 10 protein nitrogen units per mil- liliter of grass- or ragweed-pollen extract (Greer Labora- tories, Lenoir, N.C.) were selected for the study after their informed consent was obtained. Seven of the eight subjects exhibited a reponse of >lO mm induration 4 to 8 hours after intradermal injection of 100 PNU/ml of antigen ex- tract. In all eight subjects, however, prolonged antigen ad- ministration was associated with a prolonged edematous, erythematous response encompassing the 10 mm denuded blister area that was not observed at the buffer-challenge sites.
~~ce~~~~ of skin chambers and ntigen challenges
Skin chambers were attached over the denuded bases of 1 cm diameter skin blisters induced by the application of heat and suction ta the skin on the volar surface of both forearms.’ All challenges were performed after a 5-minute exposure of the surface to PBS to reduce the concentration of any mediators generated by the blister induction. Cham- bers were challenged in pairs with 500 PNU/ml of pollen antigen or PBS, respectively, for 1 hour, aspirated dry, and refilled with either 100 PNU/ml of antigen or PBS (buffer). This procedure was repeated at the second, third, fifth, seventh, and ninth hours. Fluids were centrifuged, and the supernatants for mediator assay were frozen at - 20” C for histamine and at - 70” C under nitrogen for PAF and LTB,. The cell pellet was collected as previously described, and the cells were counted in a Coulter counter (Coulter Elec- tronics, Hialeah, Fla.).’ Differential cell staining indicated that >9§% of cells assessed were neutrophilic granulocytes.
edia~~~ assays
Histamine. Histamine concentrations were measured by a rad~~enz~rnati~ technique. ’
Pm. PAF in unpurified samples was quantified by a rabbit platelt-aggregation bioassay’” that included creatine phos- phate, creatine phosphokinase, and indomethacin (Sigma Chemical Co., St. Louis, Mo.) additives to inhibit platelet aggregation by adenosine diphosphate or arachidonic acid.
Forty microliters of each chamber fluid was added to an aggregometer cuvette containing platelets, and the aggre- gation response was observed for at Ieast 2 minutes. If aggregation did not occur, a known quantity of PAF was added to the platelets to verify their capacity to aggregate. In every case, exogenous PAF elicited the expected aggre- gation response, indicating the samples without detectable PAF did not contain inhibitors of platelet aggregation. All fluids that aggregated platelets were tested for susceptibility to inhibition by the specific PAF antagoni:;ts ~adsuren~ne (Merck Sharp & Dohme, Rahway, N.J.) and BN 52021 (Institut Henri Beaufour, Le Plessis Robinson, France) to confirm that aggregation was due to PAF.13 Selected fluids, containing large quantities of PAF activity sufficient for chromatographic characterization, were further analyzed by HPLC on a Microporasil column (Waters Associates, Bed- ford, Mass.) that was developed at 1.5 ml/mm with acetonitrile, methanol, and phosphoric acid ( 130 : 5 : 15, vol/vol) to confirm the identity of the mediator. One-half milliliter fractions were collected and pooled according to the elution times of the chromatography standards. The pooled fractions were extracted twice with chloroform methanol (2: 1 vol/vol), dried, resuspended in PBS con- taming 0.1 gm/dl of human albumin, and assayed for PAF activity. The standards used and the range of elu- tion times (n = 20) were dip~~toylph~s~hatidyl~h~l~~e (Sigma Chemical Co., St. Louis, MO.), 9 to 11 minutes; [‘Hllyso-PAF, 11 to 13 minutes; lysophosphatidyl~h~l~ne~ 15 to 17 minutes; [3H]PAF (I-O-[alkyl-1’2’ HI-2-acetyl- sn-glycero-3-phosphorylcholine) (New England Nuclear, Boston, Mass.), 21 to 22 minutes; and s~hingornye~~~ (Sigma Chemical Co.), 21 to 22 minutes. Elution times of the nonradioactive standards were determined by optical absorbancy at 203 nm. Elution times and recovery of radio- labeled PAF and lyso-PAF, which have low optical absor- bancy, were determined by lipid scin~llati~n counting of aliquots of column fractions. Recovery of radiolabeled PAF exceeded 90% .I4
LTB, and oxidative products of LTB,. The concentrations of LTB, in unpurified fluids and of LTB,, 20-OH LTB,, and 20-COOH LTB, in HPLC eluates were determined by RIA with a rabbit anti-LTB, serum, [‘H]LTB, (Amersham Corp., Arlington Heights, Ill.), and synthetic LTB, 20-OH LTB,, and 20-COOH LTB, for three separate standard curves (J. Rokach, Merck Frosst, Canada, Ltd., Dorval, Canada) as described.‘* The lowest limit of reliable quantification and the mean amount required for 50% inhibition of binding of [“HILTB, were 0.03 and 0.15 pmollml for LTB,, 0.3 and 3 pmol/ml for 20-OH LTB,, and 0.3 and 3 pmoliml for 20-COOH LTB,. LTB, and the oxidation products were iso- lated from selected fluids with sufficient quantities by ex- traction on Sep-Pak octadecylsilane cartridges (Waters As- sociates, Milford, Mass.) and resolution cm a 4.5 mm by 25 cm Ultrasil reverse-phase column of 10 pm par- ticle diameter octadecylsilane (Beckman Instruments Inc. _ Palo Alto, Calif.) that was developed at 1 mllmin with methanol, 0.03 gmidl phosphoric acid in water (65:35, vol/vol) (pH 5.5 with NH,OH). The times of elution of 20-COOH LTB,, 20-OH LTB,, and LTB, were 3.7 to 3.9,
VOLUME 83
NUMBER 3
I, PAF concentrations in chamber fluids collected from antigen-cba~ienge sites
4.3 to 4.6, and 11.8 to 12.3 minutes, respectively (range, n = 5). The portions of eluate containing each compound were pooled, processed, and subjected to RIA. Recovery was estimated from that of radiolabeled compounds that had been added to portions of chamber Auids before extraction and HPLC.
All atopic volunteers challenged with pollen antigen developed a local cutaneous reaction manifested by intense erythema and swelling around the blister bases, from the second through the ninth hour. The mean (t SEM) concentration of histamine released into chamber fluids at sites of antigen stimulation at- tained a peak of 106 f 18 rig/ml at 1 hour after challenge. Histamine levels decreased during the sec- ond hour at 25 + 8 rig/ml and decreased further to a plateau of 11 to 13 ng/ ml throughout the subsequent 7 hours of continuous antigen stimulation. Histamine concentrations of 2 2 2 ng / ml in control and buffer site chamber fluids at 1 hour were significantly lower (p < 0,005) than at antigen sites. No histamine was detected at buffer sites after the first hour.
PAF was released into the chamber fluids after an- tigen stimulation in six of the eight subjects. The range of PAF concentrations in all fluids was 1 pm to 90 pmol (Tabie I). In four subjects the PAF concentration was highest at 5 to 9 hours and it was highest in two subjects at 3 to 5 hours, but all subjects had detectable
F activity at 5 to 7 hours. Chamber fluids recovered before 3 hours did not contain measurable amounts of PAF. Furthermore, none of the chamber fluids from buffer-control sites had detectable levels of PAF at anytime during the 9-hour challenge period.
Subsequent studies were designed to determine if the chamber fluid PAF is identical to the phospholipid PAF composed of 1-O-alkyl-2-acetyl-sn-glycero-3- phosphorylcholine (acetyl glyceryl ether phosphoryl-
choline or PAF acether). Each sample that contained PAF activity was tested for inhibition by selective antagonists. Platelets in aggregometer cuvettes pretreated with 5 pmol/L of kadsure~o~e or 10 pmol/L of BN 52021 for 1 minute and then chal- lenged with the chamber fluid. Every sample that had PAF activity was completely inhibited by kad- surenone. In addition, BN 5202 1 completely in three of three fluids tested with this antagonist fluids that contained large quantities of PAF activity were subjected to HPLC, and the elution time of the platelet-aggregating activity was determined. In all three samples, a single peak of PAF activity was re- covered, and the peak had a retention time cal to that of synthetic 1-O-alkyl-2-acetyl-sn-gl -3- phosphorylcholine. The activity of the PAF recovered from HPLC was also completely inhibiter surenone.
LTB, immunoreactivity was detected at antigen sites at all times during the 9 hours in the five subjects studied but achieved the highest concen~~tions in all subjects at 2 to 7 hours (Table II). Peak conce~trat~~~s of LTB, at antigen sites ranged from 6 to 36 nmol, and 38% to 80% of the amounts recovered were o-oxidation metabolites of LTB4, as RIA after HPLC. There was no co~elat~o~ between either the quantity or time of maximal conce~t~atioR of LTB, and those of PAF. In the three of five subjects from whom enough fluid was available, only low levels of LTB, (PO. 1 to 1.3 pmol/ml) were detectable at the buffer sites during the 9-hour period.
The number of cells recovered from chamber- antigen challenge and buffer-control sites for each of the time periods after the second hour are presented in Table III. As in previous studies, no cells were recovered from chamber fluids until after the third hour, and 95% of all cells recovered at any time period were neutrophilic leukocytes43 5 The number of neu- trophils at antigen sites exceeded the number at buffer
Shalit et al. J. ALLERGY CLIN. I~MU~~L.
MARCH 1989
a concentrations in chamber fluids recovered from a~tigen~chal~enge sites
ND, Not determined. “LTB, concentrations are expressed in picomoles per milliliter, and the percentage of o-oxidation metabolites of LTB, are presented in
parentheses.
. Cells recovered from chamber fluids
Antigen sites Buffer sites Time after challenge (hr) Time after challenge (hr)
Mean 9.4t 4.7 8.4 19.2t 1.2 4 2.9 8.2 + SEM 1.7 1.9 2.9 2.7 0.24 1.5 0.8 1.9
ND, Not done. *Number af leukocytes ( X 10’). lp < 0.05 compared to buffer site.
sites at all time periods but was significantly increased only at 3 to 5 hours and when all the neutrophils at each individual collection point were summed to cal- culate the total number. The data reflect in part the small number of subjects studied, as well as the vari- ability in number of cells recovered at each time period at the antigen sites in these subjects. There was no correlation between the total number of neutrophils recovered and the levels of PAF or LTB, at any time period or for the entire 9-hour period.
The late appearance of elevated levels of PAF and LTB, in buman skin chamber fluids was elicited spe- cifically by repeated intense antigen stimulation. Clearly, the patterns of release of PAF and LTB, dif- fered from the pattern of histamine. After antigen challenge and mast cell degranulation, mast pre- formed mediator histamine was released in the first
hour. No PAF was found in the first hour, an LTB, concentrations also were not maximal. During the subsequent hours after challenge with pollen an- tigen, histamine concentrations dropped to a plateau when concentrations of PAF and LTB, increased to maximal levels at 3 to 9 and 2 to 7 hours, respectively.
The lack of correlation between the amount of PAP and/or LTB, and either histamine or number of cells recovered from the chamber fluids could r either the small number of subjects studied or the lexity of the ongoing in vivo allergic reaction and release of these lipid mediators. We think the latter is most likely. The release of PAP and LTB, from cells has been demonstrated in in vitro studies to be influenced by the interaction of more than one stimulus. In neu- trophils LTB, production from diverse stimuli, such as N-formyl-methionyl-leucyl-phenylalanine, iono- phore, or opsonized zymosan can be enhanced by interactions with endotoxin and lipopolysacch~i~~~~
PAF and leukotriene B, in skin res
stim.ulated monoeytes.‘5-18 Similarly, PAF production eutrophils may be retained intracellularly unless neutrophil is acted on by other stimuli, such as toxin or C5a.l’ Thus, even if the neutrophils re-
covered from the reaction site were the source of the PAF and LTB, in the chamber fluids, the quantities of mediator-generated cells may be related to the degree of activation of the cells and interaction with other products at the reaction site rather than their number alone. Indeed, with this skin chamber model, we have demonstrated that not all cells recovered from denuded sites are equally activated, as assessed by a sensitive assay of intracellular oxidative metabolism.4 PAF is also produced by other cells, including endothelial cells ,20 mast cells ,” alveolar macrophages ,** plate- lets,23 monocytes,24 and eosinophils.25 Cell-associated PAF was produced after anti-IgE challenge of dis- ersed purified human lung mast cells, but none was
released into the supernatant.21 No conclusive evi- dence for PAF production by human cutaneous mast cells or basophils is yet at hand.
The predominant 5-lipoxygenase metabolite of ar- achidonic acid in neutrophils is LTB,.*’ Neutrophils also contain an LTB,-20-hydroxylase that converts LTB, to 20-OH LTB, and then 20-COOH LTB4.26 The neutrophils present at both allergen and buffer sites throughout the 9-hour period may be the principal source of LTB, and the o-oxidation products that we detected in the chamber fluids. The greater number of neutrophils attracted by pollen antigen challenge may account for the higher levels of LTB4 measured at these sites. High levels of LTB, have been found in other neutrophil-dominant inflammatory exudates as we1Lz7 In a previous study, using a l-hour incu- bation with antigen followed by 4 hours of replace- ment with buffer, we noted that LTC, was the pre- dominant leukotriene present in antigen-site skin chambers.’ Although we did detect low levels of LTB, (up to 1.6 pmol/ml), they were not as significant as we found in this study. These differences can be ex- plained both by the greater antigen sensitivity in our current study subjects (total histamine release of 46 ng versus 23 ng in the previous study during a similar 4-hour time period) and the more prolonged antigenic stimulus (9 hours versus 1 hour) in this study. Re- cently, Sissan et a1.28 found that PAF synthesis by human neutrophils correlated well with LTB, produc- tion. l%F is a putative allergic and inflammatory me- diator that has been detected in only a few human allergic states. Cirandel et al.” observed increased plasma levels of PAF associated with histamine in plasma of patients with primary acquired cold urticaria a few minutes after a positive challenge. Friedman et al.” detected PAF in nasal washings after intranasal
challenge with specific antigen. Indirect evidence for PAF activity during bronchoconstriction was reported by Knauer et al.” who observed platelet activation and the appearance of platelet factor 4 in plasma after positive antigen-induced bronchoprovo~ati~~. investigators3’ were not able to demo~s~ate release of PAF or platelet factor 4 in arterial plasma before, during, or after early or late asthmatic responses in humans. Of particular relevance to our sin-cb~ber model are a number of studies of the effects of PAF in the skin of humans. Injection of 30 ng per site of PAF in the skin of humans results in an early whcal and flare and a late-response (persisting up to 24 hours).g The response was dose related, not duplicated by the biologically inactive precursor lyso-PAF, and synergistically enhanced when PAF is mixed with prostaglandin &.8, “3 31 Biopsy specimens of lesions produced by intracutaneous administration of PAF re- vealed a neutrophil infiltrate, as well as lymphocytes and histocytes, during the next 30 minutes to 4 hours.’ By 32 hours, a vasculitis was evident with vascular destruction, intense infiltration with neutro- phils and mononuclear cells, and endothelial cell swelling.’
Our observation that PAF appears after antigen challenge during the time period of the late cutaneous skin reaction, in association with local skin erythema and swelling, may support the h~po~esis that this potent mediator is playing a role in the pathoge~esis of late-phase allergic reactions. It has been previously demonstrated that intradermal injection of FAF into human skin induced a reaction grossly and histopath- ologically similar to late-phase responses.’ It is con- ceivable that inflammatory cells emigrating initially to the sites of acute allergic skin reactions produce and release lipid inflammatory mediators, such as LTB4, that attract more leukocytes to the area and further stimulate them. During the subsequent hours, monocytes , eosinophils , neutrophils, and endothelial cells could generate sufficient PAF to cont~bute to further cell activation, increased vascular permeabil- ity, and recruitment of more cells, in short, simulating the tissue inflammatory changes observed during late- phase reactions.
We thank Mrs. Carolyn von Allmen, Ms. Mary Valen- zano, and Mr. Thomas Potter for their expert technical as- sistance.
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